Optical interrogation system and method for using same

ABSTRACT

An optical interrogation system and a method are described herein that enable the interrogation of one or more biosensors which can be located within the wells of a microplate. In one embodiment, the optical interrogation system has a tunable laser, N-fiber launches, N-lenses and N-detectors that are set-up to interrogate N-biosensors. In another embodiment, the optical interrogation system has a tunable laser, N-fiber launches, N+1 lenses and N-detectors that are set-up to interrogate N-biosensors.

RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.11/888,495, entitled “Optical Interrogation System And Method For UsingSame”, filed on Aug. 1, 2007, still pending.

TECHNICAL FIELD

The present invention relates to an optical interrogation system and amethod for interrogating one or more biosensors which can be locatedwithin the wells of a microplate.

BACKGROUND

Today non-contact optical sensor technology is used in many areas ofbiological research to help perform increasingly sensitive andtime-constrained assays. In one application, an optical interrogationsystem can be used to monitor changes in the refractive index orvariations in the optical response/optical resonance of an opticalbiosensor as a biological substance is brought into a sensing region ofthe biosensor. The presence of the biological substance alters theoptical resonance of the biosensor when it causes a bio-chemicalinteraction like material binding, adsorption etc. . . . It is thisalteration of the optical resonance that enables one to use thebiosensor to directly monitor a biological event in label-free assays.Examples of biosensors include surface plasmon resonance (SPR) sensorsand waveguide grating coupler (WGC) sensors. A detailed discussion aboutthe structure and function of the WGC sensor is provided in thefollowing documents:

-   -   U.S. Pat. No. 4,815,843 entitled “Optical Sensor for Selective        Detection of Substances and/or for the Detection of Refractive        Index Changes in Gaseous, Liquid, Solid and Porous Samples”.    -   K. Tiefenthaler et al. “Integrated Optical Switches and Gas        Sensors” Opt. Lett. 10, No. 4, April 1984, pp. 137-139.    -   Ph. M. Nellen, K Tiefenthaler, W. Lukosz, “Integrated Optical        Input Grating Couplers as Biochemical Sensors” Sensors and        Actuators, 15, 273 (1988).        The contents of these documents are incorporated by reference        herein.

The optical interrogation system used today to interrogate the biosensorcan take many forms, and two of the more general forms are brieflydescribed next. In one case, the optical interrogation system delivers asingle-wavelength, high-angular content optical beam to the biosensor,and the output beam received from the biosensor provides someinformation about the angular response of the biosensor. This type ofoptical interrogation system is commonly referred to as an angularinterrogation system since angular detection is employed to locate adominant angle in the output beam which is indicative of the particularoptical response/optical resonance of the biosensor. In another case,the optical interrogation system delivers a collimated optical beamcontaining a range of wavelengths to the biosensor, the output beamreceived from the biosensor provides some information about thewavelength response of the biosensor. This type of optical interrogationsystem is commonly referred to as a spectral interrogation system sincethe spectrum of the output beam is analyzed to locate the resonantwavelength in the output beam which is indicative of the particularoptical response/optical resonance of the biosensor.

These types of optical interrogation systems work relatively well butthere is still a desire to try and design a new and improved opticalinterrogation system that can be used to interrogate a biosensor todetermine if a biomolecular binding event (e.g., binding of a drug to aprotein) or if some other event occurred on a surface of the biosensor.Accordingly, there has been and is a need for a new and improved opticalinterrogation system that can be used to interrogate a biosensor. Thisneed and other needs have been addressed by the optical interrogationsystem and interrogation method of the present invention.

SUMMARY

The present invention includes an optical interrogation system and amethod for using the same to interrogate one or more biosensors. In thisembodiment, the optical interrogation system includes: (a) a tunablelaser for generating an optical beam; (b) a first fiber launch forreceiving the optical beam generated by the tunable laser and thenoutputting a first optical beam; (c) a first lens for collimating thefirst optical beam outputted from the first fiber launch such that thecollimated optical beam illuminates a first biosensor; (d) the firstlens for receiving a first reflected optical beam from the firstbiosensor; (e) a first detector, located ahead of or past a focal pointof said first lens, for receiving a first defocused optical beam fromthe first lens; and (f) a data processing device for receiving andprocessing one or more intensity spot patterns associated with the firstdefocused optical beam received at the first detector. In a preferredembodiment, this optical interrogation system would have the tunablelaser, N-fiber launches, N-lenses and N-detectors that are configured tointerrogate N-biosensors.

In another aspect, the present invention includes an opticalinterrogation system and a method for using the same to interrogate oneor more biosensors. In this embodiment, the optical interrogation systemincludes: (a) a tunable laser for emitting an optical beam; (b) a firstfiber launch for receiving the optical beam emitted from the tunablelaser and then outputting a first optical beam; (c) a first lens forcollimating the first optical beam outputted from the first fiber launchsuch that the collimated first optical beam illuminates a firstbiosensor; (d) a second lens for receiving a first reflected opticalbeam from the first biosensor; (e) a first detector for receiving thefirst reflected optical beam from the second lens; and (f) a dataprocessing device for receiving and processing one or more imagesassociated with the first reflected optical beam received at the firstdetector. In a preferred embodiment, this optical interrogation systemwould have the tunable laser, N-fiber launches, N+1 lenses andN-detectors that are configured to interrogate N-biosensors.

Additional aspects of the invention will be set forth, in part, in thedetailed description, figures and any claims which follow, and in partwill be derived from the detailed description, or can be learned bypractice of the invention. It is to be understood that both theforegoing general description and the following detailed description areexemplary and explanatory only and are not restrictive of the inventionas disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete understanding of the present invention may be had byreference to the following detailed description when taken inconjunction with the accompanying drawings wherein:

FIG. 1 is a block diagram of an exemplary optical interrogation systemwhich is configured to interrogate one or more biosensors in accordancewith a first embodiment of the present invention;

FIGS. 2-9 illustrate a block diagram, graphs and photos which are usedto help explain the results of testing an exemplary opticalinterrogation system which is configured in accordance with the firstembodiment of the present invention; and

FIG. 10 is a block diagram of an exemplary optical interrogation systemwhich is configured to interrogate one or more biosensors in accordancewith a second embodiment of the present invention.

DETAILED DESCRIPTION

Referring to FIG. 1, there is a block diagram illustrating the basiccomponents of an exemplary optical interrogation system 100 which isconfigured to interrogate one or more biosensors 102 a, 102 b . . . 102n in accordance with a first embodiment of the present invention. Theexemplary optical interrogation system 100 includes a tunable laser 104,a 1×4 splitter 106, a power tracking device 108, a wavelength trackingdevice 110, a 1×N splitter 112, N-fiber launches 114 a, 114 b . . . 114n (e.g., N-single mode optical fibers 114 a, 114 b . . . 114 n),N-lenses 116 a, 116 b . . . 116 n (e.g., N-doublets 116 a, 116 b . . .116 n) and N-detectors 118 a, 118 b . . . 118 n (e.g., N-dual detectors118 a, 118 b . . . 118 n) and a data processing device 120 (e.g.,computer 120). In this example, the biosensors 102 a, 102 b . . . 102 nare shown located within the wells 122 of a microplate 124 (note: themicroplate 124 can have any number of wells 122 such as for example96-wells, 384-wells and 1536-wells). For a detailed discussion about anexemplary microplate 124, reference is made to the co-assigned U.S.patent application Ser. No. 11/489,173 (the contents of which areincorporated by reference herein).

The tunable laser 104 (e.g., swept wavelength tunable laser 104) emitsan optical beam 126 which has a predetermined sequence of distinctwavelengths over a predetermined time period. For instance, the tunablelaser 104 can have a tuning range where the emitted optical beam 126sequences through 840 nm to 852 nm at 12 nm increments without mode hopat a tuning speed of 0.1 nm/sec to 300 nm/sec. The tunable laser 104 isshown emitting the optical beam 126 into a fiber optic cable 128 whichis connected to the 1×4 splitter 106. In this example, the 1×4 splitter106 receives the optical beam 126 and inputs the optical beam 126 intofour separate fiber optic cables 130 a, 130 b, 130 c and 130 d.

The first fiber optic cable 130 a interfaces with the power trackingdevice 108 which functions to track the changing power of the opticalbeam 126 emitted from the tunable laser 104 (note: the power of thetunable laser 104 varies as it changes the wavelength of the opticalbeam 126). The power tracking device 108 monitors this changing powerwhich is used by the data processing device 120 as a power reference toremove the effect of power variations in the output of the spectra fromthe interrogated biosensor(s) 102 a, 102 b . . . 102 n.

The second and third fiber optic cables 130 b and 130 c interface withthe wavelength tracking device 110 which functions to track the changingwavelengths of the optical beam 126 emitted from the tunable laser 104.In one embodiment, the wavelength tracking device 114 includes a fiberMach-Zehnder interferometer 132 and an athermalized etalon 134. Thefiber Mach-Zehnder interferometer 132 is used to decode theinstantaneous wavelength of the optical beam 126 to a very highresolution while operating the tunable laser 104. The athermalizedetalon 134 is used to provide an accurate reference of the wavelength ofthe optical beam 126 while operating the tunable laser 104. Analternative embodiment for tracking the changing wavelengths of theoptical beam 126 that could also be used herein was discussed in aco-assigned U.S. patent application Ser. No. 11/711,207—the contents ofwhich are hereby incorporated by reference herein).

The fourth fiber optic cable 130 d interfaces with the 1×N splitter 112which interfaces with N-fiber launches 114 a, 114 b . . . 114 n (e.g., Nsingle-mode fibers 114 a, 114 b . . . 114 n) where each fiber launch 114a, 114 b . . . 114 n outputs an optical beam 136 a, 136 b . . . 136 ntowards a corresponding lens 116 a, 116 b . . . 116 n (e.g.,collimating/imaging doublet 116 a, 116 b . . . 116 n). The lenses 116 a,116 b . . . 116 n respectively collimate the optical beams 136 a, 136 b. . . 136 n and direct the collimated optical beams 138 a, 138 b . . .138 n to illuminate and preferably overfill the corresponding biosensors102 a, 102 b . . . 102 n. In addition, the lenses 116 a, 116 b . . . 116n respectively receive the optical beams 140 a, 140 b . . . 140 n thatare reflected from the corresponding biosensors 102 a, 102 b . . . 102n. The lenses 116 a, 116 b . . . 116 n respectively focus the reflectedoptical beams 140 a, 140 b . . . 140 n which are then allowed to divergebeyond the focal points 142 a, 142 b . . . 142 c of the lenses 116 a,116 b . . . 116 n before the defocused optical beams 144 a, 144 b . . .144 n are incident upon the corresponding detectors 118 a, 118 b . . .118 n. Alternatively, the detectors 118 a, 118 b . . . 118 n could beplaced ahead of the focal points 142 a, 142 b . . . 142 n so they canreceive the defocused optical beams 144 a, 144 b . . . 144 n.

The detectors 118 a, 118 b . . . 118 n generate a sequence of intensityspot patterns 146 a, 146 b . . . 146 n from the respective biosensors102 a, 102 b . . . 102 n where each of the intensity spot patterns 146a, 146 b . . . 146 n corresponds with one of the distinct wavelengths ofthe optical beam 126 that was emitted from the tunable laser 104. Forinstance, detector 118 a generates a sequence of intensity spot patterns146 a of the illuminated biosensor 102 a where each intensity spotpattern 146 a corresponds with one of the distinct wavelengths of theoptical beam 126 (and optical beam 136 a) that was emitted from thetunable laser 104 (and the fiber launch 114 a). Lastly, the dataprocessing device 120 (e.g., computer 120) receives the collectedintensity spot patterns 146 a, 146 b . . . 146 n and has a processor 121which uses instructions accessed from memory 123 to process thecollected images 146 a, 146 b . . . 146 n to determine for examplewhether or not there was a biochemical interaction or some other eventthat occurred on one or more of the biosensors 102 a, 102 b . . . 102 n.

In the next section, an experimental optical interrogation system 100 isdescribed which is configured to be a dual defocused opticalinterrogation system 100. This discussion is followed by a discussionabout the characterization of the experimental defocused dual opticalinterrogation system 100 in terms of measuring source/detector noise,lateral and angular sensitivity, parasitic fringes and wavelength noise.In addition to these measurements, a modification to the biosensors 102a. 102 b . . . 102 n was made and experimentally evaluated and found tohave enhanced the performance of the experimental defocused dual opticalinterrogation system 100. Finally, the results of the measurements thatare associated with the testing of the experimental defocused dualoptical interrogation system 100 are summarized.

Experimental Optical Interrogation System 100

A defocused dual optical interrogation system 100 has been made andtested which had a configuration similar to the system illustrated FIG.1 except that the detectors 118 a, 118 b . . . 118 n are dual detectors118 a, 118 b . . . 118 n where each are used to receive intensity spotpatterns of both a sample region 202 and a reference region 204 on therespective biosensors 102 a, 102 b . . . 102 n (see FIG. 2). The lightsource for the experimental defocused dual optical interrogation system100 was a New Focus (6300 Velocity) 840 nm tunable laser 104 which wascoupled to a single-mode fiber output 128. The usable laser tuning rangewas approximately 12 nm between 840 nm-852 nm. The output from thetunable laser 104 was split by a 1×4 splitter 106 and input into foursingle-mode fibers 130 a, 130 b, 130 c and 130 d. The first output issent to a power tracking device 108 which was used to monitor the powerof the tunable laser 104. The second output was coupled to a fiberMach-Zehnder interferometer 132 which had an 8 pm period, while thethird output was sent to a temperature stable (ULE) etalon 134 which hada free spectral range of 188 pm. The two devices 132 and 134 where usedto monitor the wavelength of the optical beam 126 generated by thetunable laser 104. The fourth single-mode output was coupled to apolarization scrambler (Fiberpro PS 3000) which was in turn coupled tothe N-fiber launches 114 a, 114 b . . . 114 n where the correspondingoptical beams 136 a, 136 b . . . 136 n exit at the end of a flatpolished ferrules (note: the polarization scrambler was not shown inFIG. 1). The emitted light 136 a, 136 b . . . 136 n was respectivelycollimated by doublets 116 a, 116 b . . . 116 n (Thorlabs part #AC127-019-B) which had 19 mm focal lengths. The doublets 116 a, 116 b .. . 116 n produced beams 4.1 mm in diameter (to the 1/e² points) whichsubstantially overfilled the 2×2 mm biosensors 102 a, 102 b . . . 102 n.The biosensors 102 a, 102 b . . . 102 n had single gratings in which the“left half” and the “right half” respectively corresponded to the signaland reference regions 202 and 204 (see FIG. 2 which illustrates onedoublet 116 a, one biosensor 102 a and one dual detector 118 a). Thelight 140 a, 140 b . . . 140 n reflected by both “halves” of thebiosensors 102 a, 102 b . . . 102 n was focused by the respectivedoublets 116 a, 116 b . . . 116 n and then allowed to diverge beyond thefocal points 142 a, 142 b . . . 142 n of the doublets 116 a, 116 b . . .116 c before the expanded defocused beams 144 a, 144 b . . . 144 n whereincident upon the dual detectors 118 a, 118 b . . . 118 n (Hamamatsudetector 118). In this setup, the vertical distance between the fiberlaunches 114 a, 114 b . . . 114 n and intensity spot pattern planes was4 mm, between the fiber launches 114 a, 114 b . . . 114 n and lenses 116a, 116 b . . . 116 n was 19 mm and between the lenses 116 a, 116 b . . .116 n and the microplate 1024 was 12 mm.

As can be seen in FIG. 2, the light 140 a reflected from the sensor andreferences regions 202 and 204 on the biosensor 102 a respectivelyilluminated specific regions 206 and 208 on the dual detector 118 a. Fora detailed discussion about an exemplary biosensor 102 which has asample region 202 (signal spectra 302) and a reference region 204(reference spectra 304) reference is made to co-assigned U.S. patentapplication Ser. No. 10/027,509 filed on Dec. 29, 2004 and entitled“Method for Creating a Reference Region and a Sample Region on aBiosensor and the Resulting Biosensor”. The contents of this particulardocument are hereby incorporated by reference herein.

In this scheme, the sample and reference region 202 and 204 havenominally the same resonance wavelength (but are spatially resolved bythe dual detector 118 a) as has been illustrated in FIG. 3. The locationof the resonance feature, λ_(c), for the signal spectra 302 (sampleregion 202) and the reference spectra 304 (reference region 204) wasdetermined using a 1^(st) moment centroid algorithm:

$\lambda_{c} = {\sum\limits_{i = 1}^{N}\frac{\lambda_{i}\left( {P_{i} - P_{th}} \right)}{\left( {P_{i} - P_{th}} \right)}}$

where λ_(i), P_(i) are the wavelengths and corresponding power levelsabove the threshold power, P_(th).

The various sensitivity measurements (translation measurements, angularmeasurements) discussed below are referenced measurements, i.e. theywhere made by calculating the difference between the resonantwavelengths of the signal and reference spectra 302 and 304. Plus, thetranslation measurements where made by moving the microplate 124 in thex-direction (parallel to the grating lines on the biosensors 102 a, 102b . . . 102 n) and y-direction (perpendicular to the grating lines onthe biosensors 102 a, 102 b . . . 102 n) with a Newport controller (ESP300) and linear stages. This allowed precise automated measurements tobe made of the biosensors 102 a, 102 b . . . 102 b located within thewells 122 of the microplate 124. And, the angular measurements wheremade manually by using a tip-tilt stage attached to the microplate 124.The precision of the angular measurements in this particular set-up wason the order of 100 μRd.

Measurements and Calculations I. Source/Detector Noise.

The source/detector noise was measured by monitoring the differencebetween the centroids of the sample and reference regions 202 and 204 ofa given biosensor 102. In FIG. 4 there is a graph illustrating a sampleof the data (100 points) that where collected which showed a 1 sigmavalue of ˜16 fm. The data collection time for each point was ˜1 second.Similar noise values where also seen for the other biosensors 102 b . .. 102 n as long as the optical beam 138 b . . . 138 n was within 0.5 mmof the center of their respective wells 122 (and biosensors 102 b . . .102 n) within the microplate 124.

II. Translational Sensitivity

The translation sensitivity was measured over a 5×9 grid of 45 wells 122in the microplate 124 where each well 122 had their x and ytranslational dependence respectively measured. In addition, thetranslation measurements included measuring the (referenced) centroid at21 equally spaced locations spanning a length of 100 μm. For each well122, a linear fit was made to this data and then the slope of theselines where found for the x and y directions. The results of thisexperiment are shown in FIGS. 5A-5D. In particular, the translationmeasurement results for the x and y directions are respectively shown inFIGS. 5A and 5B. And, the spatial dependence of the translationalvariations in the x and y directions are respectively shown in FIGS. 5Cand 5D. In viewing FIGS. 5A-5D, it can be seen that the translationalsensitivity is about 50-100 fm/μm or less in the x-direction and is onlyabout 10-20 fm/μm in the y-direction. The values in the y-direction arebasically at the noise limit of the experimental defocused dual opticalinterrogation system 100.

Next, the translational sensitivity associated with a single well 122(biosensor 102 a) of the microplate 124 was examined. The translationalsensitivity was measured on a 7×7 grid within a single well 122(biosensor 102 a) covering an area of 1.5 mm square. The translationalmeasurement results for all 49 data points in the x-direction andy-direction are respectively shown in FIGS. 6A and 6B. And, the spatialdependence of the translational sensitivity in the x-direction andy-direction within the 1.5 mm region of the well 122 (biosensor 102 a)are respectively shown in FIGS. 6C-6D (note: that in these two figuresany point that was measured higher than 150 μm/μ was plotted as equal tothis value).

In viewing FIGS. 6A-6D, it can be seen that the lateral sensitivity inthe x direction was about 70 fm/μm or greater while the lateralsensitivity was about 20 fm/μm in the y-direction. One possiblecontributing factor for this dependency is the fact that for translationin the x-direction, the sizes of the signal and reference regions 202and 204 are altered by the x-translation of the microplate 20. In otherwords, as the microplate 124 is moved in the +x direction then thesignal region 202 (for example) will increase in size as the referenceregion 204 decreases in size. Of course, the opposite is true for xtranslation in the reverse direction. Therefore, variations in theresonance wavelength across the well 122 (biosensor 102 a) adverselycontributed to the lateral sensitivity. This is most likely the largestcontributor to the lateral sensitivity which accounts for why thedependency in the x-direction is greater than it is in the y-direction.The small dependence in the y-direction is possibly a result of the factthat the optical beam 138 a is not perfectly uniform over the well 122(biosensor 102 a) within the microplate 124. This would cause adifferent power weighting to be applied to the resonance (assuming thesensor resonant wavelength is not spatially uniform) which gives rise toa small amount of y-direction (and also x-direction) lateral dependency.For y translations, the sizes of the reference and signal regions 202and 204 are not altered.

A modification was made to the biosensors 102 a, 102 b . . . 102 n in anattempt to reduce the lateral (and angular, see next section)dependency. In particular, opaque strips/masks 210 (in this case 400 μmdiameter wires) where placed within the wells 122 to divide theindividual biosensors 102 a, 102 b . . . 102 n into two equal signal andreference regions 202 and 204. Because of the 400 μm wide opaquestrips/masks 210 on the biosensors 102 a, 102 b . . . 102 n, the size ofthe signal and reference regions 202 and 204 would not change as themicroplate 124 was moved in the x-direction for translations up to 400μm length. With the modified biosensors 102 a, 102 b . . . 102 n, thelateral sensitivity in the x-direction and the y-direction within asingle masked well 122 and three masked wells 122 where measured (in thesame manner as was done above) and the results have been displayed inFIGS. 7A-7F. In particular, the translational measurement results forall 49 data points in the x-direction and y-direction of the single well122 (having the masked biosensor 102) are respectively shown in FIGS. 7Aand 7B (note: of these 49 measured points, only those with sensitivitiesless than 150 fm/μm are shown in FIGS. 7A and 7B) And, the spatialdependence of the translational sensitivity in the x-direction andy-direction within the 1.5 mm region of the single well 122 (having themasked biosensor 102) are respectively shown in FIGS. 7C-7D (note: thatin these two figures any point that was measured higher than 150 fm/μwas plotted as equal to this value). As can be seen, with the maskedbiosensor 102 the lateral sensitivity had been significantly improved tobe approximately 20 fm/μm in both the x-direction and the y-direction.Finally, the lateral sensitivity was measured across three wells 122(with three masked biosensors 102) and the translational sensitivity inthe x-direction and y-direction for these three wells 122 have beenrespectively shown in FIGS. 7E and 7F.

III. Angular Sensitivity

The microplate holder used in the angular sensitivity test was equippedwith manual controls for tilting the microplate 124 about the x- andy-axes. For the measurements of the angular sensitivity, the microplate124 was rotated in increments of 1 mRad. Finer increments would havebeen desirable since the one sigma error on plate tilt was expected tobe ˜12 μRad. Nonetheless, the angular sensitivity did not show adramatic change within these relatively coarse adjustments, so it wasreasonable to extrapolate these results to the small tilt angles thatwould likely be encountered in an operational defocused dual opticalinterrogation system 100. In this experiment, two sets of angularsensitivity measurements were made: one for the microplate 124 withoutthe opaque masked (wire) wells 122 (biosensors 102) and one where themicroplate 124 had the opaque masked (wire) wells 122 (masked biosensors102).

For the microplate 124 without the mask, the measured dependencies fortilt in the x- and y-axes are respectively shown in FIGS. 8A and 8B. Inviewing FIGS. 8A and 8B, it can be seen that the slopes of the curves inthe graphs indicate that the angular sensitivity is about 1 fm/μRad fortilt about the x-axis and 13 fm/μRad for tilt about the y-axis. Thishigher dependence about the y-axis was expected since this tilt shiftsthe size of the signal and reference regions 202 and 204 (in a similarmanner as was described for the x-lateral translation measurements).Since, the tilt variation is expected to be 12 μRd (one sigma), thisimplies a tilt sensitivity of 12 fm about the x-axis, and a much highersensitivity of 156 fm about the y-axis of an operational defocused dualoptical interrogation system 100.

Next, the angular sensitivity measurements were repeated for themicroplate 124 with the 400 μm masked wells 122 (masked biosensors 102).The measured dependencies for tilt in the x- and y-axes for this casehave been respectively shown in FIGS. 9A and 9B. For this case, it canbe seen that the angular sensitivity was about 2 fm/μRad for tilt aboutthe x-axis and about 2 fm/μRad for tilt about the y-axis. This lowerdependence about the y-axis (over a 2 mRad range) was expected since themask 210 on the biosensor 102 maintained the size (and location) of thesignal and reference regions 202 and 204 while tilting the microplate124. Thus, for this configuration and using the expected tilt variationof 12 μRd implies that the tilt sensitivity would be only 24 fm abouteither the x-axis or y-axis for an operational defocused dual opticalinterrogation system 100.

IV. Parasitic Fringes

Multiple reflections within the microplate 124 (which in these testshappened to be 0.7 mm thick) gave rise to fringes which had an ˜0.3 nmperiod in the resulting resonance spectrum. These fringes can beproblematical since they could bias the perceived resonance peak. Forthe results that have been presented herein, these fringes have beenremoved in software (by convolving the measured resonance with a 400 pmwide gaussian) prior to performing any sort of analysis. This softwarefiltering technique is applicable here since the width of the resonancewas sufficiently wide compared with the period of these parasiticfringes. However, if an operational defocused dual optical interrogationsystem 100 is used then a digital filtering technique could be used toremove parasitic interference fringes before the final computation ofcentroid wavelengths. Examples of several digital filtering techniqueswhere discussed in the co-assigned U.S. Patent Application No.60/781,397 filed on Mar. 10, 2006 and entitled “Optimized Method for LIDBiosensor Resonance Detection”. The contents of this document are herebyincorporated by reference herein.

V. Wavelength Noise

The wavelength noise affects the accuracy with which the wavelengths ofthe data points can be measured. The value of this particular wavelengthuncertainty was not measured in these experiments since the contributionthat this noise/error makes to the measurement has already been takeninto account in the source/detector noise measurement tests. Plus, thewavelength noise has been estimated to be below 50 fm.

Experimental Conclusions

The experimental defocused dual optical interrogation system 100described herein has been evaluated for various performance parametersincluding, source/detector noise, translational/lateral sensitivity andangular sensitivity. A modification to the biosensors 102 a, 102 b . . .102 n in which a mask 210 has been employed yielded improved performanceof the experimental defocused dual optical interrogation system 100. Abrief summary of these results are provided in TABLE 1:

TABLE 1 Dual Detector Optical Interrogation System 100 Source/Detectornoise 16 fm Lateral sensitivity 25 fm/μm Angular sensitivity  2 fm/μRad

Referring to FIG. 10, there is a block diagram illustrating the basiccomponents of an exemplary optical interrogation system 1000 which isconfigured to interrogate one or more biosensors 1002 a, 1002 b . . .1002 n in accordance with a second embodiment of the present invention.The exemplary optical interrogation system 1000 includes a tunable laser1004, a 1×4 splitter 1006, a power tracking device 1008, a wavelengthtracking device 1010, a 1×N splitter 1012, N-fiber launches 1014 a, 1014b . . . 1014 n (e.g., N-single mode optical fibers 1014 a, 1014 b . . .1014 n), N+1 lenses 1016 a, 1016 b . . . 1016 n, 1016 n+1 (e.g., N+1doublets 1016 a, 1016 b . . . 1016 n, 1016 n+1) and N-detectors 1018 a,1018 b . . . 1018 n (e.g., N-dual detectors 1018 a, 1018 b . . . 1018 n)and a data processing device 1020 (e.g., computer 1020). In thisexample, the biosensors 1002 a, 1002 b . . . 1002 n are shown locatedwithin the wells 1022 of a microplate 1024 (note: the microplate 1024can have any number of wells 1022 such as for example 96-wells,384-wells and 1536-wells). For a detailed discussion about an exemplarymicroplate 1024, reference is made to the co-assigned U.S. patentapplication Ser. No. 11/489,173 (the contents of which are incorporatedby reference herein).

The tunable laser 1004 (e.g., swept wavelength tunable laser 1004) emitsan optical beam 1026 which has a predetermined sequence of distinctwavelengths over a predetermined time period. For instance, the tunablelaser 1004 can have a tuning range where the emitted optical beam 1026sequences through 840 nm to 852 nm at 12 nm increments without mode hopat a tuning speed of 0.1 nm/sec to 300 nm/sec. The tunable laser 1004 isshown emitting the optical beam 1026 into a fiber optic cable 1028 whichis connected to the 1×4 splitter 1006. In this example, the 1×4 splitter1006 receives the optical beam 1026 and inputs the optical beam 1026into four separate fiber optic cables 1030 a, 1030 b, 1030 c and 1030 d.

The first fiber optic cable 1030 a interfaces with the power trackingdevice 1008 which functions to track the changing power of the opticalbeam 1026 emitted from the tunable laser 1004 (note: the power of thetunable laser 1004 varies as it changes the wavelength of the opticalbeam 1026). The power tracking device 1008 monitors this changing powerwhich is used by the data processing device 1020 as a power reference toremove the effect of power variations in the output of the spectra fromthe interrogated biosensor(s) 1002 a, 1002 b . . . 1002 n.

The second and third fiber optic cables 1030 b and 1030 c interface withthe wavelength tracking device 1010 which functions to track thechanging wavelengths of the optical beam 1026 emitted from the tunablelaser 1004. In one embodiment, the wavelength tracking device 1010includes a fiber Mach-Zehnder interferometer 1032 and an athermalizedetalon 1034. The fiber Mach-Zehnder interferometer 1032 is used todecode the instantaneous wavelength of the optical beam 1026 to a veryhigh resolution while operating the tunable laser 1004. The athermalizedetalon 1034 is used to provide an accurate reference of the wavelengthof the optical beam 1026 while operating the tunable laser 1004. Analternative embodiment for tracking the changing wavelengths of theoptical beam 1026 that could also be used herein was discussed in theco-assigned U.S. patent application Ser. No. 11/711,207—the contents ofwhich are hereby incorporated by reference herein).

The fourth fiber optic cable 1030 d interfaces with the 1×N splitter1012 which interfaces with N-fiber launches 1014 a, 1014 b . . . 1014 n(e.g., N single-mode fibers 1014 a, 1014 b . . . 1014 n) where eachfiber launch 1014 a, 1014 b . . . 1014 n outputs an optical beam 1036 a,1036 b . . . 1036 n towards a corresponding collimating/imaging lens1016 a, 1016 b . . . 1016 n (note: the last lens 1016 n+1 is not coupledwith a fiber launch 1014)). The lenses 1016 a, 1016 b . . . 1016 nrespectively collimate the optical beams 1036 a, 1036 b . . . 1036 n anddirect the collimated optical beams 1038 a, 1038 b . . . 1038 n toilluminate and preferably overfill the corresponding biosensors 1002 a,1002 b . . . 1002 n. The adjacent lenses 1016 b, 1016 c . . . 1016 n+1receive the optical beams 1040 a, 1040 b . . . 1040 n that arerespectively reflected from the corresponding biosensors 1002 a, 1002 b. . . 1002 n (note: this configuration is different than the opticalinterrogation system 100 shown in FIG. 1 where the lenses 116 a, 116 b .. . 116 n respectively receive the optical beams 140 a, 140 b . . . 140n that are reflected from the corresponding biosensors 102 a, 102 b . .. 102 n). Then, the adjacent lenses 1016 b, 1016 c . . . 1016 n+1 focusthe corresponding reflected optical beams 1040 a, 1040 b . . . 1040 nwhich are then allowed to diverge beyond the focal points 1042 a, 1042 b. . . 1042 n of the adjacent lenses 1016 b, 1016 c . . . 116 n+1 so theoptical beams 1044 a, 1044 b . . . 1044 n form an image of thebiosensors 1002 a, 1002 b . . . 1002 n at an image plane upon thecorresponding detectors 1018 a, 1018 b . . . 1018 n (e.g., dualdetectors 1018 a, 1018 b . . . 1018 n can be used to receive the samplespectra and reference spectra from corresponding biosensors 1002 a, 1002b . . . 1002 n). Alternatively, the detectors 1018 a, 1018 b . . . 1018n can be positioned ahead or before the focal points 1042 a, 1042 b . .. 1042 n and not at the image plane and in this particular configurationthe detectors 1018 a, 1018 b . . . 1018 n would still receive images ofthe biosensors 1002 a, 1002 b . . . 1002 n.

The detectors 1018 a, 1018 b . . . 1018 n generate a sequence of images1046 a, 1046 b . . . 1046 n of the respective biosensors 1002 a, 1002 b. . . 1002 n where each of the images 1046 a, 1046 b . . . 1046 ncorresponds with one of the distinct wavelengths of the optical beam1026 that was emitted from the tunable laser 1004. For instance,detector 1018 a generates a sequence of images 1046 a of the illuminatedbiosensor 1002 a where each image 1046 a corresponds with one of thedistinct wavelengths of the optical beam 1026 (and optical beam 1036 a)that was emitted from the tunable laser 1004 (and the fiber launch 1014a). Lastly, the data processing device 1026 (e.g., computer 1026)receives the collected images 1046 a, 1046 b . . . 1046 n and has aprocessor 1021 which uses instructions accessed from memory 1023 toprocess the collected images 1046 a, 1046 b . . . 1046 n to determinefor example whether or not there was a biochemical interaction or someother event that occurred on one or more of the biosensors 1002 a, 1002b . . . 1002 n.

The exemplary optical interrogation system 1000 has a configuration thatis desirable since it effectively solves a problem where it is nowpossible to collect the optical signals 1040 a, 1040 b . . . 1040 n fromindividual biosensors 1002 a, 1002 b . . . 1002 n in the microplate 1024while enabling the detectors 1018 a, 1018 b . . . 1018 n and thecorresponding fiber launches 1014 a, 1014 b . . . 1014 n to bepositioned relatively close enough to one another. In the past, this wasa problem because the physical geometry and placement of the fiberlaunches and detectors would dictate the minimum separation possible.One potential solution to this problem involved increasing the angle ofincidence of the optical beams that were directed to the biosensors.However, the angle of incidence can only be increased to a point,otherwise vignetting will occur thereby compromising performance. Theoptical interrogation system 1000 of the present invention effectivelyaddresses this problem because it's configuration allows one to obtain alarge separation between each pair of fiber launches 1014 a, 1014 b . .. 1014 n and the detectors 1018 a, 1018 b . . . 1018 n thus one canincrease the angle of incidence of the optical beams 1036 a, 1036 b . .. 1036 n and avoid the problematical vingetting by emitting the opticalbeams 1038 a, 1038 b . . . 1038 n from one lens 1016 a, 1016 b . . .1016 n and then collecting the reflected light 1040 a, 1040 b . . . 1040n with the corresponding adjacent lens 1016 b, 1016 c . . . 1016 n+1(see FIG. 10). In addition, the reflected optical beams 1040 a, 1040 b .. . 1040 n can be detected away from both the launch plane and imageplane or at the image plane by the corresponding detectors 1018 a, 1018b . . . 1018 n. However, there are several advantages for detecting atthe image plane: (1) the spot location does not shift with the tilt (orbow) of the microplate 1024 therefore, this setup is insensitive to thisparameter; and (2) the detectors 1018 a, 1018 b . . . 1018 n are locatedat a different plane than the fiber launches 1014 a, 1014 b . . . 1014 nthereby allowing more space for the detectors 1018 a, 1018 b . . . 1018n. For example, the vertical distance between the fiber launches 1014 a,1014 b . . . 1014 n and image planes can be 7 mm, between the fiberlaunches 1014 a, 1014 b . . . 1014 n and lenses 1016 a, 1016 b . . .1016 n can be 18 mm and between the lenses 1016 a, 1016 b . . . 1016 nand the microplate 1024 can be 65 mm.

In comparing the optical interrogation system 100 and the opticalinterrogation system 1000 it should be noted that in the configurationof the optical interrogation system 100 the lenses 116 a, 116 b . . .116 n do not form a (real) image (in the classical sense) of thebiosensors 102 a, 102 b . . . 102 n for any placement location of thedetectors 118 a, 118 b . . . 118 n. In other words, the opticalinterrogation system 100 does not have an image plane beneath the lenses116 a, 116 b . . . 116 n that corresponds to the biosensors 102 a, 102 b. . . 102 n above the lenses 116 a, 116 b . . . 116 n. However, it istrue at various locations of the detectors 118 a, 118 b . . . 118 neither ahead of or past the focal points 142 a, 142 b . . . 142 n of thelenses 116 a, 116 b . . . 116 n that the detectors 118 a, 118 b . . .118 n can still “see” the biosensors 102 a, 102 b . . . 102 b (but notright at the focal points 142 a, 142 b . . . 142 n of the lenses 116 a,116 b . . . 116 n). This is because the optical interrogation system 100has a large “depth of focus” which is a result of the fact that there isonly collimated light 140 a, 140 b . . . 140 n being reflected from thebiosensors 102 a, 102 b . . . 102 n.

In contrast, the optical interrogation system 1000 has a distancebetween the lenses 1016 a, 1016 b . . . 1016 n, 1016 n+1 and themicroplate 1024 that is significantly larger than in the opticalinterrogation system 100.

Because of this increased distance the detectors 1018 a, 1018 b . . .1018 n can be located at the image plane, that is, the biosensors 1002a, 1002 b . . . 1002 n can be imaged onto the detectors 1018 a, 1018 b .. . 1018 n. Again, it should be appreciated that the detectors 1018 a,1018 b . . . 1018 n could be positioned ahead of or after the imageplane and still give a workable system (as long as the detectors 1018 a,1018 b . . . 1018 n are not located at the focal points 1042 a, 1042 b .. . 1042 n). However, placing the detectors 1018 a, 1018 b . . . 1018 nat the image plane is preferred since it results in an opticalinterrogation system 1000 that is insensitive to the curvature of themicroplate 1024. In addition, it should be appreciated that if one wereto build the optical interrogation system 100 then they could create theoptical interrogation system 1000 by adding lens 1016 n+1 (and adetector 1018 n to receive the light 1040 n from this lens 1016 n+1) andby moving the microplate 1024 “up” and “to the right” (compare FIGS. 1and 10).

Although two embodiments of the present invention have been illustratedin the accompanying Drawings and described in the foregoing DetailedDescription, it should be understood that the invention is not limitedto the embodiments disclosed, but is capable of numerous rearrangements,modifications and substitutions without departing from the spirit of theinvention as set forth and defined by the following claims.

1. An optical interrogation system comprising: a tunable laser forgenerating an optical beam; a first fiber launch for receiving theoptical beam generated by said tunable laser and then outputting a firstoptical beam; a first lens for collimating the first optical beamoutputted from said first fiber launch such that the collimated opticalbeam illuminates a first biosensor; said first lens for receiving afirst reflected optical beam from the first biosensor; a first detector,located ahead or past a focal point of said first lens, for receiving afirst defocused optical beam from said first lens; and a data processingdevice for receiving one or more intensity spot patterns associated withthe first defocused optical beam received at said first detector.
 2. Theoptical interrogation system of claim 1, further comprising: a secondfiber launch for receiving the optical beam generated by said tunablelaser and then outputting a second optical beam; a second lens forcollimating the second optical beam outputted from said second fiberlaunch such that the collimated second optical beam illuminates a secondbiosensor; said second lens for receiving a second reflected opticalbeam from the second biosensor; a second detector, located ahead or pasta focal point of said second lens, for receiving the second defocusedoptical beam from said second lens; and said data processing device forreceiving one or more intensity spot patterns associated with the seconddefocused optical beam received at said second detector.
 3. The opticalinterrogation system of claim 1, further comprising a wavelengthtracking device which tracks the distinct wavelengths of the opticalbeam generated by said tunable laser.
 4. The optical interrogationsystem of claim 1, further comprising a power tracking device whichreceives the optical beam generated by said tunable laser and tracks thepower of said tunable laser.
 5. The optical interrogation system ofclaim 1, wherein said data processing device further processes the oneor more intensity spot patterns to determine whether or not there was abiochemical interaction on the biosensor.
 6. The optical interrogationsystem of claim 1, wherein: said first biosensor is located within afirst well of a microplate; and said first biosensor has a mask locatedon a surface thereon to separate a signal region from a referenceregion.
 7. A method for interrogating one or more biosensors, saidmethod comprising the steps of: emitting a first optical beam which hasa predetermined sequence of distinct wavelengths over a predeterminedtime period; using a first lens to collimate the first emitted opticalbeam such that the first collimated optical beam illuminates a firstbiosensor; using said first lens to receive a first reflected opticalbeam from the first biosensor; using a first detector to receive thefirst reflected optical beam from said first lens, where the firstdetector is positioned ahead or past a focal point of said first lens;and using a data processing device to receive a plurality of intensityspot patterns associated with the first reflected optical beam that wasreceived at said first detector where each intensity spot patterncorresponds with one of the distinct wavelengths of the first emittedoptical beam.
 8. The method of claim 7, further comprising the steps of:emitting a second optical beam which has a predetermined sequence ofdistinct wavelengths over a predetermined time period; using a secondlens to collimate the second emitted optical beam such that the secondcollimated optical beam illuminates a second biosensor; using saidsecond lens to receive a second reflected optical beam from the secondbiosensor; using a second detector to receive the second reflectedoptical beam from said second lens, where the second detector ispositioned ahead or past a focal point of said second lens; and using adata processing device to receive a plurality of intensity spot patternsassociated with the second reflected optical beam that was received atsaid second detector where each intensity spot pattern corresponds withone of the distinct wavelengths of the second emitted optical beam. 9.The method of claim 7, further comprising a step of tracking thedistinct wavelengths of the first emitted optical beam.
 10. The methodof claim 7, further comprising a step of tracking the power of a tunablelaser which generated the first emitted optical beam.
 11. The method ofclaim 7, further comprising a step of using the data processing deviceto process the intensity spot patterns to determine whether or not therewas a biochemical interaction on the first biosensor.
 12. The method ofclaim 7, wherein: said first biosensor is located within a first well ofa microplate; and said first biosensor has a mask located on a surfacethereon to separate a signal region from a reference region.